At present, the majority of recombinant protein-based medicines are produced in mammalian cells or single cell organisms such as bacteria and yeast. However, the capital investment and operational costs associated with these systems are very high. For example, a mammalian cell-based manufacturing plant can cost upwards of $250 million. To achieve greater cost savings, and to address a capacity deficit in the global demand for recombinant protein-based pharmaceuticals, plants are being explored as alternative protein productions hosts (Giddings et al., 2000; Staub et al., 2000; Daniell et al., 2001; Walmsley et al., 2003). Different plant tissues such as leaves, seeds and tubers have been engineered for producing useful recombinant proteins (Vandekerckhove et al., 1989; Sijmons et al., 1990; Pen et al., 1992; Herbers et al., 1995; Ma et al., 1995; van Rooijen et al., 1995; Arakawa et al., 1998; Y Kusnadi et al., 1998; Zeitlin et al., 1998; Farran et al., 2002; Tackaberry et al., 1999). In a number of studies, tobacco has been used as a host plant but has some major drawbacks, including that tobacco is not a major food substance in a mammalian diet.
Granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine of clinical importance. The mature GM-CSF is a polypeptide of 127 amino acid residues (Cantrell et al., 1985; Lee et al., 1985; Wong et al., 1985) and it regulates production and function of white blood cells (granulocytes and monocytes), which are important in fighting infections (Metcalf, 1991). GM-CSF is now an integral part of the clinical management for life-threatening neutropenia, the most common toxicity of cancer chemotherapy (Dale, 2002). Other oncology applications include treatment of febrile neutropenic conditions and support following bone marrow transplantation (Dale, 2002). Potential applications are also under evaluation in patients with pneumonia, Crohn's fistulas, diabetic foot infections and a variety of other infectious conditions including HIV-related opportunistic infections (Dale, 2002). The high cost of human GM-CSF in prior culture systems has placed practical limits on its widespread use (Dale, 2002). Previously, human GM-CSF has been produced by recombinant means in COS (Wong et al., 1985), yeast (Ernst et al., 1987) and Namalwa cells (Okamoto et al., 1990). GM-CSF has also been expressed in tobacco, but at very low levels (James et al., 2000; Sardana et al., 2002).
U.S. Pat. No. 5,677,474 (Rogers) teaches a method of producing foreign polypeptides in the seeds of cereal crops, including rice. Transformation of barley plants with a GUS reporter gene is disclosed. No transgenic plants containing GM-CSF were produced.
U.S. Pat. No. 5,889,189 (Rodriguez et al.) teaches a method of producing heterologous peptides in monocots including rice. Expression of a GUS reporter gene in transgenic rice seed is disclosed. No transgenic plants containing GM-CSF were produced.
James et al. (2000) used transformed tobacco cell suspensions to produce and secrete GM-CSF, which was then isolated from the growth medium. Yields were low (maximum of 250 microgram/L) and a complicated process of adding stabilizing proteins and increasing salt concentration of the growth media was necessary to enhance recovery of secreted GM-CSF. No transgenic cereal crops containing GM-CSF were produced.
Sardana et al. (2002) disclose the production of GM-CSF in transgenic tobacco seed. Yields were low with seed extracts containing recombinant human GM-CSF protein up to a level of 0.03% of total soluble protein. No transgenic cereal crops containing GM-CSF were produced.